Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Activation of tobacco defense responses by tobacco mosaic virus (TMV) infection includes both local resistance, manifested as necrotic lesion formation resulting from host cell death at the site of infection (hypersensitive response, HR), and systemic resistance induced in the surrounding and distal uninfected parts of the plant (systemic acquired resistance, SAR) (Ryals et al., 1994; Dempsey and Klessig, 1995). Numerous studies have demonstrated that salicylic acid (SA) is an endogenous signal for the activation of several plant defense responses including synthesis of pathogenesis-related (PR) proteins (Ryals et al., 1994, Dempsey and Klessig, 1995). However, the components which transduce the signal between SA and PR genes remain to be elucidated.
Protein kinases and phosphatases play pivotal roles in regulating and coordinating many signal transduction pathways in living organisms, including but not limited to, cell division, cell differentiation, and responses to environmental stimuli (Hunter, 1995). In plants, protein phosphorylation has been implicated in responses to many signals, such as light, hormones, pathogen attack, temperature stress, and nutrient starvation (Stone and Walker, 1995). Several genes which encode putative protein kinases, including CTR1 (Kieber et al., 1993), ETR1 (Chang et al., 1993) and Pto (Martin et al., 1993) appear to participate in the transmission of signals generated by extracellular stimuli. The CTR1 and ETR1 protein kinases of Arabidopsis thaliana play a role in the ethylene signaling pathway. CTR1 is homologous to the mammalian Raf protein kinase which participates in the mitogen-activated protein (MAP) kinase cascade (Kieber et al., 1993). The Pto protein kinase from tomato, as well as its downstream kinase Pti1 which is phosphorylated by Pto, participate in conferring resistance to the pathogenic bacterium Pseudomonas syringae (Martin et al., 1993; Zhou et al., 1995).
The MAP kinase cascade is a major signaling system by which cells transduce extracellular stimuli into intracellular responses (Herskowitz 1995; Seger and Krebs, 1995; Votjek and Cooper, 1995; Kyriakis and Avruch, 1996). Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were the first of the MAP kinase family to be cloned; they are activated by diverse extracellular stimuli. Other related mammalian MAP kinases include Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPK) and p38 kinases (Seger and Krebs, 1995). MAP kinases are activated by phosphorylation on both threonine (T) and tyrosine (Y) residues within a TXY phosphorylation motif, where X can be Glu (E), Pro (P), or Gly (G). Three subfamilies of MAP kinases have been defined based on their phosphorylation motifs TEY, TPY, and TGY, which correspond to ERK1/2, JNK/SAPK, and p38 subfamilies, respectively (Kyriakis and Avruch, 1996).
The MAP kinase family is present in a diverse array of organisms including mammals, Xenopus, Drosophila, yeast, Dictyostelium, and plants. An increasing body of evidence suggests that MAP kinases play important roles in plants (Nishihama, et al., 1995). Seven MAP kinases have been identified in Arabidopsis. AtMPK1 and AtMPK2 are thought to be involved in cell proliferation (Mizoguchi, et al., 1993; Mizoguchi, et al., 1994), while AtMPK3 appears to play a role in responding to touch, cold and salinity stress (Mizoguchi et al., 1996). Several kinases upstream of the MAP kinase in the cascade have also been identified including cATMEKK1 from Arabidopsis and NPK1 from tobacco (Nishihama, et al., 1995; Mizoguchi, et al., 1996).
Several kinase activities, believed to be MAP kinases based on the fact that they preferentially phosphorylate myelin basic protein (MBP) and are themselves phosphorylated on tyrosine residues, have been shown to be activated by stress or phytohormones. These include the cutting (wounding)-induced p46 kinase (Usami et al., 1995; Seo et al., 1995), the fungal elicitor-induced p47 kinase from tobacco (Suzuki et al., 1995), and the abscisic acid-induced kinase from barley (Knetsch et al., 1996).
Protein phosphorylation/dephosphorylation events have been correlated with the activation of defense responses in a number of plants (Dietrich et al., 1990; Felix et al., 1991; Viard et al., 1994; Conrath et al., 1997). More recently, it has been shown that the activation of the potato PR-10a gene requires the phosphorylation of the nuclear factor PBF-1 (Despros et al., 1994). In addition, protein kinase inhibitors were able to block both the fungal elicitor-induced oxidative burst and the H.sub.2 O.sub.2 -mediated activation of defense genes in soybean suspension cells (Levine et al., 1994). Kinase(s) and/or phosphatase(s) also appear to regulate the activation of defense responses in tobacco, since lesion formation after TMV infection was inhibited by the phosphatase inhibitor okadaic acid (Dunigan et al., 1995). Okadaic acid also blocked the SA-mediated induction of PR-1 gene expression (Conrath et al., 1997). The kinase(s) and/or phosphatase(s) involved in these processes, however, remain to be identified.
It has been shown that the MAP kinases associated with several different signaling pathways are activated by H.sub.2 O.sub.2 or other oxidative stresses in mammalian systems. These include JNK, ERK2, and the big mitogen-activated protein kinase 1 (BMK1; Abe et al., 1996; Lo et al., 1996; Guyton et al., 1996; Sundaresan et al., 1995). Thus, H.sub.2 O.sub.2 appears to regulate a wide variety of different processes. In neutrophils, reactive oxygen species produced by the NADPH oxidase induce tyrosine phosphorylation, in addition to their role in bacterial killing (Fialko et al., 1994; Brumell et al., 1996). The prominent 42-44 kDa tyrosine-phosphorylated polypeptide induced by H.sub.2 O.sub.2 was later shown to be MAP kinase that is phosphorylated on both tyrosine and threonine by a redox-sensitive MAP kinase kinase (MEK; Fialkow et al., 1994). In plants, H.sub.2 O.sub.2 has been implicated in both the action of SA (Chen et al., 1993) and the induction of defense responses after pathogen invasion, exposure to ozone or UV light (Medhy, 1994; Levine et al., 1994; Kangasjarvi et al., 1994; Green and Fluhr, 1995).